Mechanisms of bradykinin-mediated Ca super(2+) signalling in canine cultured corneal epithelial cells

• Published in: Cellular signalling 2001-08, Vol.13 (8), p.565-574
• Main Authors: Huang, SCM, Chien, Chin-Sung, Hsiao, Li-Der, Wang, Chuan-Chwan, Chiu, Chi-Tso, Liang, Kao-Yi, Yang, Chuen-Mao
• Format: Article
• Language: English
• ISSN: 0898-6568

Experiments were designed to differentiate the mechanisms of bradykinin receptors mediating the changes in intracellular Ca super(2+) concentration ([Ca super(2+)] sub(i)) in canine cultured corneal epithelial cells (CECs). Bradykinin and Lys-bradykinin caused an initial transient peak of [Ca super(2+)] sub(i) in a concentration-dependent manner, with half-maximal stimulation (pEC sub(50)) obtained at 6.9 and 7.1, respectively. Pretreatment of CECs with pertussis toxin (PTX) or cholera toxin (CTX) for 24 h did not affect the bradykinin-induced [Ca super(2+)] sub(i) changes. Application of Ca super(2+) channel blockers, diltiazem and Ni super(2+), inhibited the bradykinin-induced Ca super(2+) mobilization, indicating that Ca super(2+) influx was required for the bradykinin-induced responses. Addition of thapsigargin (TG), which is known to deplete intracellular Ca super(2+) stores, transiently increased [Ca super(2+)] sub(i) in Ca super(2+) -free buffer, and subsequently induced Ca super(2+) influx when Ca super(2+) was readded to this buffer. Pretreatment of CECs with TG completely abolished bradykinin-induced initial transient [Ca super(2+)] sub(i), but had slight effect on bradykinin-induced Ca super(2+) influx. Pretreatment of CECs with 1-[ beta -[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl]-1H-imidazole (SKF96365) and 1-(6-((17 beta -3-methoxyestra- 1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione (U73122) inhibited the bradykinin-induced Ca super(2+) release and Ca super(2+) influx, consistent with the inhibition of receptor-gated Ca super(2+) channels and phospholipase C (PLC) in CECs, respectively. These results demonstrate that bradykinin directly stimulates B sub(2) receptors and subsequently Ca super(2+) mobilization via a PTX-insensitive G protein in canine CECs. These results suggest that bradykinin-induced Ca super(2+) influx into the cells is not due to depletion of these Ca super(2+) stores, as prior depletion of these pools by TG has no effect on the bradykinin-induced Ca super(2+) influx that is dependent on extracellular Ca super(2+) in CECs.